Diarrheal is a leading cause of death and illness throughout the world and causes 560 million deaths of children under 5 years old. Rotavirus (RV) causes about 70% of viral diarrheas and even with two vaccines kills 215,000 children yearly. RV diarrhea is mainly due to excess Cl- secretion that drives fluid loss, but the cellular pathways responsible for RV-induced fluid secretion are not well defined, which limits efforts to develop and testing of potential anti-diarrheal drugs that can be used to treat children when protection by vaccines is insufficient. Activation of chloride (Cl-) secretory channels is regulated by intracellular calcium (Ca2+) and cyclic nucleotide (cAMP) signals These signals in turn activate Ca2+-activated Cl- channels (CaCC), such as anoctamin (Ano) family, and cAMP-activated Cl- channels (cAMP-CC), such as the cystic fibrosis transmembrane regulator (CFTR). RV diarrhea is the classic example of Cl- secretion due to elevated Ca2+ because a hallmark of RV infection is increased Ca2+ signaling, which is induced by the RV nonstructural protein 4 (NSP4). Ano channels are the prominent CaCCs in the gut; however, the Cl- channels responsible for RV diarrhea have yet to be identified. Additionally, NSP4 is considered the primary initiator of RV-induced Ca2+ signals; however, the mechanisms by which NSP4-derived signals cause Cl- channel activation and fluid secretion have not been adequately explored. In Aim 1, we will identify the Cl- channels responsible for RV diarrhea using both in vitro and in vivo approaches. Little is known about the expression of different Cl- channels throughout the human intestine, particularly children, we will use human intestinal enteroids (HIEs) to determine gene expression of the key Cl- channels and their regulatory proteins. Next, we will measure the ability of RV to activate individual Cl- channels using a fluorescence quench assay and determine whether those channels contribute to fluid secretion using the enteroid swelling assay (ESA) in the presence of specific Cl- channel blockers. Lastly, we will determine whether inhibitors of Ano1 or CFTR will attenuate diarrhea and if so whether intestinal specific deletion of those channels also reduces diarrhea. In Aim 2, we will determine whether RV NSP4 activates both Ca2+ and cAMP through activation of the sensor stromal interacting molecule 1 (STIM1). STIM1 activation can generate Ca2+ signals through the store-operated Ca2+ entry (SOCE) pathway, as well as cAMP signals through the store-operated cAMP signaling (SOcAMPs) pathway. We will use live cell imaging of intestinal cell lines and HIEs expressing fluorescent Ca2+ and cAMP biosensors to determine whether RV and NSP4 activate both SOCE and SOcAMPs and use the ESA to determine whether the Ca2+/cAMP signals generated by NSP4 cause fluid secretion. Lastly, we will determine whether blockers of SOCE or SOcAMPs will attenuate RV diarrhea in vivo. Our results will generate critical insights into the mechanisms of viral diarrhea and new biosensor HIE model systems that can be applied to future studies enteric virus, such as noroviruses and astroviruses.